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South African Journal of Animal Science 2013, 43 (No.


Germination as a processing technique for soybeans in small-scale farming

N.C. Kayembe# & C. Jansen van Rensburg

Department of Animal and Wildlife Sciences, University of Pretoria, Pretoria 0002, South Africa

(Received 4 December 2013; Accepted 6 June 2013; First published online 15 June 2013)

Copyright resides with the authors in terms of the Creative Commons Attribution 2.5 South African Licence.
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Animal Science.

Heat processing is an effective way of reducing antinutritional factors (ANFs) in legumes, but requires
expensive facilities and equipment. Accurate control of temperature is critical to avoid under- or overheating.
Therefore, heat treatment of soybeans is not a viable option for the average small-scale farmer in remote
areas. Germination and other traditional methods, namely soaking and dehulling, were evaluated as
alternative processing methods for soybeans. The effect of the processing treatment on the level of different
ANFs, nutritional composition and in vitro protein digestibility (IVPD) of soybean seeds was determined.
Soybeans were soaked for 24 hours and allowed to germinate for one to six days. Soaked soybeans were
manually dehulled. Changes within seeds were noted for nutritional and ANFs during germination. Crude
protein and fat content increased with increasing number of days germinated, but germination caused a
decrease in starch content. Dehulling also increased the crude protein and fat content of the seeds. All
treatments caused a significant decrease in condensed tannins of the soybeans. Trypsin inhibitor levels were
numerically lower after germinating, soaking and dehulling of seeds, but not significantly so. There were no
changes in IVPD after treatment of the soybeans. It was concluded that germination for a period of three
days effectively improved the nutritional value of soybeans and can be considered an alternative treatment of
soybeans for the small-scale farmer where heat treatment is impractical or impossible.

Keywords: Soybeans seed, Glycine max, TIA, anti-nutritional factors, traditional methods
Corresponding author: ndonda.kayembe@gmail.com

Legume seeds are important sources of energy and protein in many parts of the world, both in animal
and human nutrition (Kaushik et al., 2010). However, their nutritional value may be compromised in part by
the presence of undesirable components, known as antinutritional factors (ANFs) (Donangelo et al., 1995).
Processing legumes with heat is a quick technique for decreasing or destroying ANFs, but strict
guidelines must be applied for the treatment to be effective. An optimal processing temperature must be
applied, as overheating can cause protein and amino acid damage. On the other hand, underprocessing does
not completely destroy ANFs (Kabeya & Kiatoko, 2004). Expensive facilities and equipment are required for
the proper heat treatment of legumes, putting this technique out of reach of the average small-scale farmer in
remote areas.
Globally, soybean (Glycine max) seed is one of the largest sources of vegetable seed oil and protein in
the feed and food industry, and contains about 40% crude protein and 20% oil. It is also a source of calcium,
iron, carotene and ascorbic acid. Soybean oilcake meal has become the principal protein supplement for
livestock in many countries (Ghaly & Sutherland, 1982) and is one of the legumes that is most frequently
used in the poultry industry. However, soybeans contain undesirable components such as lipoxygenase and
trypsin inhibitors that limit their utilisation. Trypsin inhibitor is an ANF that affects protein digestibility
(Kumar et al., 2006). A high level of trypsin inhibitors in a diet stimulates pancreatic juice secretion and

URL: http://www.sasas.co.za
ISSN 0375-1589 (print), ISSN 222-4062 (online)
Publisher: South African Society for Animal Science http://dx.doi.org/10.4314/sajas.v43i2.7
168 Kayembe, 2013. S. Afr. J. Anim. Sci. vol. 43

causes pancreatic hypertrophy and poor growth in animals (Liener & Kakade, 1980, cited by Huisman &
Tolman, 2001).
Traditional processing methods of soybeans such as germinating, soaking and dehulling are sometimes
used to reduce or eliminate the ANFs that affect protein utilisation. The trial reported here was designed to
evaluate the efficacy of germination and other traditional treatments as alternative processing methods in
reducing the levels of ANFs in soybean seed.

Materials and Methods

One ton of soybean seeds (variety RR5409 Monsanto) was procured locally (Brits, North West
Province, South Africa). The seeds were kept raw or subjected to one of four processing methods: roasted,
soaked for one or two days, dehulled or germinated from one to six days. The methods of processing were as
As a heat-treated control group, a representative sample of seed was roasted in an oven for 30 min at
130 C. After cooling, the seeds were milled and stored in plastic bottles at room temperature for
further analysis.
For the soaking treatment, 500 g representative soybean samples were weighed into 5000 mL plastic
containers, which were then filled with tap water. The seeds were soaked at room temperature for
24 or 48 h. The soaked seeds were spread on hessian sacks for 48 to 72 h under sunlight until dry.
After drying, the seeds were milled through a 1 mm sieve and kept in plastic containers at room
temperature for further analysis.
The soybean seeds were dehulled after being soaked for 24 h in tap water (1 : 10, w/v). The hulls
were removed manually by rubbing the seeds between the palms of the hands (Sangronis, 2007).
After dehulling, the hulls were removed by flotation, and the cotyledons were dried under sunlight.
The dehulled seeds were milled through a 1 mm sieve, and then stored at room temperature, pending
further analysis.
Germination was achieved by soaking the seeds for 24 h. Afterwards they were spread indoors on
hessian sacks on the floor, and covered with aluminium foil to exclude light. Six groups of seeds
were kept at room temperature (22 - 26 C) and allowed to germinate for one to six days,
respectively. Water was applied once daily to provide moisture during sprouting. Thereafter, the
germinated beans were dried for 48 to 72 h under sunlight, ground through a 1 mm sieve, and then
kept at room temperature pending further analysis.
The sampling of seeds was carefully conducted to ensure homogenous and representative samples for
analysis. Large samples were collected from various locations from the original batch of beans, and were
properly mixed, divided and subdivided until the required sample size was obtained.
All treatments were replicated four times, independent of one another. Representative samples from
each treatment from all four batches were analysed in duplicate for moisture (AOAC, 2000), crude protein
(AOAC, 2000), starch (Davie, 1986), condensed tannins (Porter et al., 1986), total polyphenols (Makkar,
1993), trypsin inhibitor activity (TIA) (Kakade et al., 1974), crude fat (AOAC, 2000) and in vitro protein
digestibility. Analyses were done at the UP-Nutrilab (Department of Animal and Wildlife Sciences,
University of Pretoria, South Africa).
In vitro protein digestibility (IVPD) of the seed was determined by pepsin or papain enzyme digestion.
For the pepsin method, the soybeans (300 g) were weighed into a series of test tubes. A solution of 5 mL of
0.075 N HCl and 0.5 mL of pepsin solution (2.0 mg/mL) in 0.075 N HCl was added to each tube. The tubes
were incubated at 37 C and enzyme action was stopped after 24 h by adding 5 mL 10% (w/v) trichloroacetic
acid (TCA). Digestion was performed in duplicate. The digest was filtered through Whatman No. 2 filter
paper, and the residue was washed with warm water on the filter. Nitrogen in the residue was estimated by
the micro-Kjeldahl method. The IVPD was obtained by calculating the difference between the amount of
total nitrogen in the sample before and after in vitro digestion with pepsin (AOAC, 1965). Kjeldahl nitrogen
was multiplied by the factor 6.25 to obtain crude protein.
In vitro protein digestibility with papain was conducted according to the method used by Buchanan &
Byers (1969) and modified by Nanda et al. (1977). The activator solution of 0.5 M thioglycolic acid was
prepared by adding 4.6 g (3.5 mL) laboratory-grade thioglycolic acid to distilled water and making it up to
100 mL, after having adjusted the pH to 6.6 with a 0.45 M NaOH solution. A phosphate-citrate buffer
Kayembe, 2013. S. Afr. J. Anim. Sci. vol. 43 169

solution of pH 6.6 was made by mixing 7.7 parts 0.2 M Na2HPO4 with 2.3 parts 0.1 M citric acid. Quantities
of substrate representing about 10 mg of protein N were weighed accurately in 75 mL centrifuge tubes. Then
0.4 mL papain solution was pipetted into each digestion tube, together with 0.5 mL 0.5 M thioglycolic acid
and 9.1 mL citrate-phosphate buffer. Two drops of toluene were added as preservative, the tubes were
stoppered, and the contents were mixed gently. Care was taken to ensure that all particles of the protein
sample remained immersed in the digestion liquid. The samples were incubated in duplicate at 37 C for
24 h. The control tubes contained only enzyme, activator and buffer. Immediately after incubation, the
unhydrolyzed protein fraction was precipitated by adding 10 mL trichloroacetic acid (TCA) solution (100
g/L) to each tube. Thereafter the procedure was followed as described for pepsin.
An analysis of variance with the GLM model (SAS, 2010) was used to determine the significance
between treatments. Mean and standard deviations were calculated. Treatment means were separated using
Fishers protected least significant difference (LSD) at the 5% level of significance.

Results & Discussions

Soybeans were germinated for various time periods, from 1 to 6 days, in order to find the optimal
germination period. Raw and roasted soybeans were used as negative and positive controls, respectively.
Soaked and dehulled soybeans were also assessed for their nutritional value, levels of ANFs and in vitro
protein digestibility.
The germinated soybeans contained less (P <0.05) starch compared with all other treatments,
including the raw beans (Table 1). Mubarak (2005) reported a decrease in starch content in mung bean seeds
after three days of germination. Mwikya et al. (2000) also found a gradual decrease in the starch content of
finger millet during the first 36 h of germination. This reduction was attributed to the hydrolysis of starch
that supplies the developing seedling with energy during germination (Madden et al., 1985).
Crude protein was found to increase as the number of days of germination of soybeans increased
(Table 1) and was higher (P <0.05) than the crude protein content of beans from the raw control group and
the roasted and soaked treatments. Kaushik et al. (2010) also reported an increase in crude protein with
germination of soybeans. Similar increases in protein have been reported for other legumes such as lablab
beans (Osman, 2007), mung beans (Mubarak, 2005), faba beans and kidney beans (Alonso et al., 1998). The
apparent increase in protein can be attributed to the utilization of carbohydrates as an energy source for
developing sprouts (Donangelo et al., 1995). However, Bamdad et al. (2009) reported a notable decrease in
protein content after lentils germinated for 1, 3 and 5 days. Echendu et al. (2009) found a release of free
amino acids after enzymatic hydrolysis for the synthesis of new protein.

Table 1 Effect of soaking, dehulling and germination of soybean seeds on crude protein, fat and starch

Treatment Crude protein Crude fat Starch

Raw 39.1a 0.37 15.8a 1.34 1.24ab 0.11

Roasted 40.3b 0.57 19.6b 1.03 1.38a 0.20
Soaking 1 day 40.2b 0.10 17.7b 0.82 1.17b 0.11
Soaking 2 day 42.1c 0.25 19.0c 0.40 0.96c 0.03
Dehulling 45.1d 0.35 21.0d 0.54 1.11bc 0.12
Germination 1 day 41.3e 0.19 18.1d 0.89 0.99d 0.89
Germination 2 day 42.2f 0.54 19.1e 1.37 0.84d 0.12
Germination 3 day 43.4g 0.20 19.2ef 0.99 0.75d 0.10
Germination 4 day 43.8g 0.52 19.2e 1.05 0.71d 0.13
Germination 5 day 44.1gh 0.70 18.5e 0.97 0.94d 0.20
Germination 6 day 45.1di 0.30 20.6f 0.60 0.86d 0.24

Mean ( standard deviation) values within the same column with different superscripts differ significantly at P <0.05.
170 Kayembe, 2013. S. Afr. J. Anim. Sci. vol. 43

Germination of the soybeans increased the fat contents significantly (P <0.05) (Table 1). This agrees
with Echendu et al.s (2009) report on ground beans. However, Ghavidel & Prakash (2007) found a
significant decrease of fat content after germination of some legume seeds. Osman (2007) and Mubarak
(2005) reported a significant decrease of fat content when mung been seeds were allowed to germinate for
three days. This could be owing to the use of fat as energy during the sprouting process. The other processing
methods that were included in this study, that is, soaking, dehulling and roasting, led to significant increases
of crude protein and fat content. These increases are owing to the removal of the hull portion and therefore
the concentration of endosperm in the legume product (Ghavidel & Prakash, 2007) as protein and fat are
characteristically present in the cotyledon fraction of seeds (Alonso et al., 1998).
Table 2 shows the effect of the different treatments on the levels of various ANFs. The time of
germination and the duration of soaking were important factors affecting the ANFs measured in the study.
All treatments numerically decreased the activity of trypsin inhibitor measured in the soybeans. These
differences did not prove to be statistically significant, probably because of large variations found among
replicates. However, germination may be useful in removing or reducing the activity of trypsin inhibitors in
Germination significantly reduced the levels of condensed tannins in the soybeans. Condensed
tannins were significantly decreased by 46%, 55% and 54%, respectively, after 2, 3 and 4 days of
germination. This reduction in tannin content in soybeans is attributed to the tannin activity during
germination (Mubarak, 2005) that leads to the formation of hydrophobic association of tannins with seed
proteins and enzymes. Some loss of condensed tannins during germination may be because of the leaching of
tannins into water. Reddy et al. (1985) found that overnight soaking of grains followed by germination for
two days significantly reduced the tannin content by 50% in a wide variety of legumes. The decrease in
condensed tannins after soaking, as observed in this study, is in agreement with results reported by Alonso
et al. (2000).
Dehulling decreased the levels of condensed tannins, similarly to the findings of Alonso et al. (1998).
Tannins are located mainly in the seed coats, which could explain their reduction after dehulling (Matthus
& Angelini, 2003).

Table 2 Effect of soaking, dehulling and germination of soybeans on trypsin inhibitor activity (mg/g), total
polyphenols (%) and condensed tannins (%)

Treatment Trypsin inhibitor activity Total polyphenols Condensed tannins

Raw 83.1 8.71 10.8a 0.42 1.24a 0.34

Roasted 52.3 14.55 12.4b 1.07 1.50a 0.46
Soaking 1 day 77.3 9.67 12.4ab 0.20 1.04ab 0.62
Soaking 2 day 66.2 5.31 12.4ab 1.56 0.71c 0.21
Dehulling 68.7 9.20 12.8ab 1.35 0.64c 0.18
Germination 1 day 64.0 24.97 11.8ab 0.24 0.82c 0.29
Germination 2 day 67.9 21.54 11.2ab 0.77 0.67c 0.14
Germination 3 day 64.4 18.86 10.6ab 0.69 0.56c 0.22
Germination 4 day 69.0 8.35 10.4ab 0.70 0.57c 0.28
Germination 5 day 63.9 6.67 12.4bc 1.41 0.96c 0.39
Germination 6 day 67.3 10.77 12.8ac 0.69 0.87c 0.26

Mean ( standard deviation) values within the same column with different superscripts differ significantly at P <0.05.

In this study, the germination of soybean seeds did not have a significant effect on the level of total
polyphenols in the beans. The increase of total polyphenols observed in the soybeans that were germinated
for a period of five to six days could be attributed to the presence of fungi at this time (Wu et al., 2011), as
Kayembe, 2013. S. Afr. J. Anim. Sci. vol. 43 171

fungal growth on the seeds was evident from the fifth day of germination. Further research is needed before
definite conclusions can be made.
Various researchers have reported lower levels of tannins, TIA and other ANFs in pulses as a result of
domestic processing, including soaking, dehulling and germinating. Mwikya et al. (2000) found that the TIA
decreased threefold, and reported a significant decrease in tannins to undetectable levels in finger millet
during sprouting. A 20% - 38% reduction in tannins was observed during the germination of green grams,
cowpeas, lentils and chickpeas (Ghavidel & Prakash, 2007). Sangronis & Machado (2007) found a
significant decrease of TIA in pigeon beans, white beans and black beans after five days of germination,
while Frias et al. (1995) found that six days of germination of lentils decreased TIA. Wang et al. (1997)
reported that TIA decreased with shorter germination periods, but increased slightly as the length of
germination increased. In this study, the level of total polyphenols increased after five days of germination,
and also after roasting, soaking and dehulling soybeans (Table 2). Changes in phenolic compounds may
occur, depending on the types of seed (Lpez-Amors et al., 2006), processing conditions such as rinsing
(Frias, 1995), presence of light, and germination time (Frias, 1995; Oloyo, 2004; Lpez-Amors et al., 2006;
Dueas et al., 2009). Oloyo, (2004) and Dueas et al. (2009) found that polyphenols of lupin increased
during germination. Similarly, Satwadhar et al. (1981) found that the polyphenol levels in moth beans
increased from 1.0% to 1.7% at 36 h germination, while beans that germinated for 48 h had higher amounts
of polyphenols than raw moth beans.
No significant differences were noted in IVPD among the treatments, regardless of the type of enzyme
used (Table 3). On the contrary, Mubarak (2005) reported that dehulling, soaking and germinating mung
bean seeds improved their IVPD. A significant increase was observed in the IVPD of white beans, black
beans and pigeon beans after germination (Alonso et al., 2000; Sangronis & Machado, 2007).

Table 3 Effect of soaking, dehulling and germination of soybeans on in vitro protein digestibility (%)

Treatment Pepsin digestion Papain digestion

Raw 66.5 21.40 62.7 13.01

Roasted 44.6 19.79 58.4 2.77
Soaking 1 day 65.4 24.44 55.4 11.13
Soaking 2 day 80.0 7.03 57.2 16.58
Dehulling 71.9 24.69 48.0 9.01
Germination 1 day 64.6 14.02 55.4 9.77
Germination 2 day 63.8 15.96 52.6 8.90
Germination 3 day 59.8 19.38 52.5 8.90
Germination 4 day 57.8 15.15 49.5 17.99
Germination 5 day 58.1 14.36 46.8 9.79
Germination 6 day 62.8 11.47 50.7 4.56

Mean ( SD) reported on dry basis.

All traditional processing methods tested in this study significantly increased the crude protein and fat
content of the soybeans, but decreased the starch content. Germination for a period of three to four days was
effective in improving their nutritional value and reducing the levels of condensed tannins. Trypsin inhibitor
activities were numerically lower in all treated soybeans, but not significantly. No statistical differences were
noted between the soybeans that were germinated for a period of three days and those that were germinated
for four days. Furthermore, longer periods of germination may result in fungal growth that may be
detrimental to the quality of the soybeans. In this study, notable fungal growth after five days of germination
might have contributed to an increase in the polyphenol content of the beans. It is recommended that small-
172 Kayembe, 2013. S. Afr. J. Anim. Sci. vol. 43

scale farmers in rural areas apply a germination period of three days for soybeans when properly controlled
heat treatment is not possible. The cost of germinating soybeans is minimal as expensive equipment and
specialised facilities are not necessary. Because soybeans are used mostly in poultry diets, the findings of the
study are probably most applicable in the field of poultry nutrition. However, in vivo studies on poultry are
needed to substantiate the efficacy of germination as a processing method for soya.

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